Composition for an injectable bone mineral substitute material

ABSTRACT

The invention refers to an injectable composition for a bone mineral substitute material, which comprises a dry powder mixed with an aqueous liquid. The powder comprises a first reaction component comprising a calcium suphate hemihydrate with the capability of being hardened to calcium sulphate dihydrate when reacting with said aqueous liquid; a second reaction component, which comprises a calcium phosphate with the capability of being hardened to a calcium phosphate cement when reacting with said aqueous liquid; and at least one accelerator for the reaction of said first and/or second reaction component with said aqueous liquid. A method of producing an injectable bone mineral substitute material is also provided, wherein the composition is mixed in a closed mixing and delivery system for delivery.

TECHNICAL FIELD

The present invention relates to an injectable composition for a bonemineral substitute material with the capability of being hardened in abody fluid in vivo. Furthermore, the invention relates to a method ofproducing such a material.

BACKGROUND ART

During the last decade, the number of fractures related to osteoporosis,i.e. reduced bone mass and changes in microstructure leading to anincreased risk of bone fractures, has almost doubled. Due to thecontinuously increasing average life time it is estimated that by 2020people over 60 years of age will represent 25% of Europe's populationand that 40% of all women over 50 years of age will suffer from anosteoporotic fracture.

With the aim to reduce or eliminate the need for bone grafting, researchhas been made to find a suitable artificial bone mineral substitute.Presently, at least the following bone mineral substitutes are used forthe healing of bone defects and bone fractures, namely calciumsulphates, as for instance Plaster of Paris, calcium phosphates, as forinstance hydroxylapatite, and polymers, as for instancepolmethylmetacrylate (PMMA).

Calcium sulphate (Plaster of Paris), CaSO₄·1/2H₂O, was one of the firstmaterials investigated as a substitute for bone grafts. Studies havebeen undertaken since 1892 to demonstrate its acceptance by the tissuesand rapid rate of resorbtion. It has been concluded that Plaster ofParis implanted in areas of subperiosteal bone produces no furtheruntoward reaction in the tissue than normally is present in a fracture.Regeneration of bone in the area of subperiosteal resection occursearlier than when an autogenous graft is used. Plaster of Paris does notstimulate osteogenesis in the absence of bone periosteum. The new bonegrowing into Plaster of Paris is normal bone. No side effectsattributable to the implantation of Plaster of Paris have been noted inadjacent tissues or in distant organs. However, Plaster of Paris has thedrawback of very long setting times, which constitutes problems atsurgery.

Another group of materials for substituting bone tissue in fracturesites and other bone defects is calcium phosphate cements. Due to theirbiocompatibility and their osteoconductivity they can be used for bonereplacement and augmentation.

Hydroxylapatite, a crystalline substance which is the primary componentof bone, is mainly used as a bone substitute, but is not strong enoughfor use under weight bearing conditions. Experiments have shown thathydroxylapatite cement forms a stable implant in respect of shape andvolume over 12 months and has the same excellent tissue compatibility asexhibited by commercial ceramic hydroxylapatite preparations.Microscopic examination clearly demonstrated that hydroxylapatite cementwas progressively ingrown by new bone over time.

Although the ideal is to achieve hydroxylapatite, there are alsoapatite-like calcium phosphates which can be obtained as potential bonesubstitutes. In Table 1 calcium phosphates are presented which areformed by a spontaneous precipitation at room or body temperature, aswell as the pH range, within which these components are stable.

TABLE 1 Calcium phosphates obtained by precipitation at room or bodytemperature Ca/P Formula Name pH 0.5 Ca(H₂PO₄)•H₂O MCPM 0.0-2.0 1CaHPO₄•2H₂O DCPD 2.0-6.0 1.33 Ca₈(HPO₄)₂,(PO₄)₄•5H₂O OCP 5.5-7.0 1.5Ca₉(HPO₄)(PO₄)₅OH CDHA 6.5-9.5 1.67 Ca₅(PO₄)₃OH PHA 9.-5-12

Other calcium phosphates can be obtained by means of sinteringtemperatures, above 1000° C. (Table 2). These calcium phosphates can notbe obtained by precipitation in room or body temperature. However, theycan be mixed with an aqueous solution alone or in combinations withother calcium phosphates to form a cement-like paste which will set withtime.

TABLE 2 Components forming calcium phosphate cements Ca/P CompoundFormula Name 1.5 α-tricalcium phosphate α-Ca₃(PO₄)₂ α-TCP 1.5β-tricalcium β-Ca₃(PO₄)₂ β-TCP 1.67 Sintered hydroxylapatiteCa₁₀(PO₄)₆(OH)₂ SHA 2.0 Tetracalcium phosphate Ca₄(PO₄)₂O TTCP

Bone mineral substitute materials can be used for preparing a pastewhich can be injected directly into a fracture site. The paste isinjected into the void in the bone and, hardening, an implant isobtained which conforms to the contours of the gap and supports thecancellous bone. Both calcium sulphate and hydroxylapatite materialshave been extensively investigated as a possible alternative toautogenous bone grafts to help restore osseous defects of bone andfixation of bone fracture.

In this connection it is important that a complete stability is obtainedas quickly as possible during or after surgery in order to preventmotions at site of healing. This especially applies to fractures, butalso when filling of a bone cavity or replacing bone lost during tumorremoval the healing is inhibited by movements and the in-growth of newbone is prevented.

It is also of importance that the hardened material is so similar instructure to the bone so that it can be gradually resorbed by the bodyand replaced by new bone growth. This process can be facilitated if thehardened cement is provided with pores, which can transport nutrientsand provide growth sites for new bone formation.

M. Bohner et al. disclosed at the Sixth World Biomaterials CongressTransactions (15-20/5 2000) a method to obtain an open macroporouscalcium phosphate block by using an emulsion of a hydrophobic lipid(oil) in an aqueous calcium phosphate cement paste or an emulsion of anaqueous calcium phosphate cement paste in oil. After setting, the cementblock was sintered at 1250° C. for 4 hours. Likewise, CN 1193614 shows aporous calcium phosphate bone cement for repairing human hard tissue.The cement contains pore-forming agent which maybe a non-toxicsurfactant, or a non-toxic slightly soluble salt, acidic salt andalkaline salt.

Studies have also been made on mixtures of the above mentioned bonemineral substitute materials. In U.S. Pat. No. 4,619,655 is disclosed abone mineral substitute material comprising a mixture of Plaster ofParis, i.e. calcium sulphate hemihydrate, and calcium phosphate ceramicparticles, preferably composed of hydroxylapatite, or tricalciumphosphate or mixtures thereof. According to U.S. Pat. No. 4,619,655tests show that when alloplasts composed of 50/50 mixtures ofhydroxylapatite/Plaster of Paris were implanted into experimentallycreated defects in rat mandible, the Plaster of Paris was completelyresorbed within a few weeks and replaced by connective tissue. Thehydroxylapatite was not resorbed and some particles were eventuallycompletely surrounded by bone. It was therefore concluded that thePlaster of Paris acted as a scaffold for the incorporation ofhydroxylapatite into bone.

A recent study presented on the “Combined Orthopaedic Research SocietiesMeeting”, Sep. 28-30, 1998, Hamamatsu, Japan, also shows additionaltests relating to mixtures of Plaster of Paris and hydroxylapatite.According to this study a combination of hydroxylapatite particles andPlaster of Paris had a viscosity which allowed an easy placement of theimplant material and prevented migration of hydroxylapatite particlesinto surrounding tissues during and after implantation. The experimentsshowed that Plaster of Paris was absorbed in relatively short time, waseasily manipulated with hydroxylapatite particles, and did not interferewith the process of bone healing.

WO 9100252 shows a composition which is capable of hardening in bloodwithin about 10-45 min. The composition comprises essentially calciumsulphate hemihydrate with small amounts of calcium sulphate dihydrate.Organic and inorganic materials, such as hydroxylapatite, can also beincluded in the composition. After hardening, particles ofhydroxylapatite are obtained within a calcium sulphate cement. Thecalcium sulphate cement is dissolved rapidly by aqueous body fluidswithin four weeks, leaving solid particles of hydroxylapatite.

Likewise, such particles of hydroxylapatite within a calcium sulphatecement are obtained by the method of WO 9117722. The composition for useas an animal implant comprises calcium sulphate hemihydrate, calciumphosphate, and sodium sulphate. The calcium phosphate is hydroxylapatiteand the sodium sulphate enables the composition to be used in thepresence of blood or other body fluids.

SUMMARY OF THE INVENTION

The object of the invention is to provide an injectable composition fora bone mineral substitute material with the capability of being hardenedin a body fluid in vivo, which hardens during surgery with accompanyingearly control of fracture fragment movement as well as provides a stablelasting implant over a year with high mechanical strength, and whichduring this later period presents a porous as well as irregularstructure for bone in growth.

A further object of the present invention is to provide such an improvedinjectable bone mineral substitute for filling defects in osteoporoticbone and for additional fracture fixation in substantially cancellousbone which does not exhibit the drawbacks of high viscosity at deliveryand low fracture toughness.

Still another object of the invention is to provide an injectable bonemineral substitute having excellent biocompatibility, favorablebiological and rheological properties. The bone mineral substituteshould also be biodegradable and be possible to sterilize by radiationor gas without suffering a significant deterioration in properties.

In order to achieve these objects the injectable composition accordingto the invention has been given the characterizing features of claim 1.

According to the invention a composition is provided which comprises twotypes of bone cement materials, which both are subjected to a hardeningreaction in contact with water.

A cement of hardened calcium sulphate (gypsum) will remain set in a dryenvironment. In a wet environment, such as in a Body Simulated Solution,this material will immediately start to disintegrate. Thus, an implantedmaterial with reduced strength will be obtained in the body. The solidmaterial obtained will start to degrade, eventually within 1-2 days.

On the other hand, in order to induce a setting (hardening) reaction ina Body Simulated Solution or in a body with its blood, saline can beused. By using saline a setting will be obtained immediately under anyconditions, but the implant obtained will still degrade quite rapidly.

The second reaction, in which a calcium phosphate is hardened (cemented)to a calcium phosphate cement in the presence of water, will take longertime—about 18 h or more—in order to set to a high strength material.During this period of time the already set sulphate will confer aninitial strength to the implant, and when the setting reaction oftricalcium phosphate to a high strength material is completed, a finalstrength will be obtained, which lasts for months or years.

In this connection the term “calcium phosphate cement” refers to therecognized definition (S. E. Gruninger, C. Siew, L. C Chow, A. O'Young,N. K. Tsao, W. B. Brown, J. Dent. Res. 63 (1984) 200) of a reactionproduct of a powder or a mixture of powders which—after mixing withwater or an aqueous solution to a paste—at a temperature around roomtemperature or body temperature react with the formation of aprecipitate, which contains crystals of one or more calcium phosphatesand which sets by the entanglement of the crystals within theprecipitate. Thus, different calcium phosphate products (calciumphosphate cements) can be obtained during the setting reaction independence on the component(s) of the powders used for the pasteinventive injectable composition for a bone mineral substitute material.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be explained in more detail, reference being madeto the accompanying drawings, in which

FIG. 1 shows the effects of a-tricalcium phosphate on compressivestrength;

FIG. 2 shows the effects of the content of calcium sulphate dehydrate onthe injection time; and

FIG. 3 shows the effects of the water content and the content of calciumsulphate dehydrate on the setting time.

In order to accomplish an injectable bone mineral substitute materialhaving improved characteristics, tests were made with the object toevaluate the effects of particle size, water content and accelerator onthe viscosity, setting time and porosity of the injectable bone mineralsubstitute material of the invention.

The inventive injectable composition for a bone mineral substitutematerial comprises a dry powder mixed with an aqueous liquid. A mainrequirement on such a material is its setting time, which should bewithin 5-12 minutes. Additionally, the viscosity of the material shouldbe adapted to render it injectable into the bone for 1-5 minutes afterthe beginning of the mixing procedure.

The evaluated materials comprised calcium sulphate hemihydrate, alsoknown as Plaster of Paris. It was found that the addition of a smallamount of finely ground already reacted calcium sulphate dihydrate,CaSO₄·2H₂O, had a decisive impact on the setting time and the injectabletime of the bone mineral substitute. Due to the addition of anaccelerator the setting time period was considerably shortened while theinjectable time was still long enough to make it possible to inject thematerial of the invention into e.g. a bone cavity. It is assumed thatother accelerators and mixtures of accelerators may be used, e.g.starch, mixtures of calcium sulphate. dihydrate and lignosulphate,calcium sulphate dihydrates having composite coatings, etc.

Those reactions which forms hydroxylapatite, i.e. precipitatedhydroxylapatite (PHA) or calcium deficient hydroxylapatite (CDHA), canbe classified into three groups. The first group consists of calciumphosphates, which are transformed into hydroxylapatite by a hydrolysisprocess in an aqueous solution (eq. 1-5).

5Ca (H₂PO₄)·H₂O→Ca₅(PO₄)₃OH+7H₃PO₄+4H₂O   (1)

5CaHPO₄·2H₂O→Ca₅(PO4)3OH+2H₃PO₄+9H₂O   (2)

5Ca₈H₂(PO₄)₆·5H₂O→8Ca₅(PO₄)₃OH+6H₃PO₄+17H₂O   (3)

5Ca₃(PO₄)₂+3H₂O →3Ca₅(PO₄)₃OH+H₃PO₄   (4)

3Ca4(PO₄)₂O +3H₂O→2Ca₅(PO₄)₃OH+Ca(OH)₂   (5)

Precipitated hydroxylapatite is the least soluble calcium phosphate atpH over 4.2. This means that any other calcium phosphate present in anaqueous solution at this pH range will tend to dissolve, with theprecipitation of PHA as a product. This hydrolysis process(Ca(OH)₂-H₃PO₄-H₂O) is very slow due to a decrease in supersaturation asthe reaction proceeds.

The only calcium phosphate which can react via a hydrolysis process toan apatite without the formation of sub-products is a-tricalciumphosphate (eq. 6), and the apatite formed in this reaction is a calciumdeficient hydroxylapatite.

3α−Ca₃ (PO₄)₂+H₂O→Ca₉(HPO₄) (PO₄)₅OH   (6)

The second group of reactions to a hydroxylapatite, i.e. precipitatedhydroxylapatite (PHA) or calcium deficient hydroxylapatite (CDHA), isthe combinations between TTCP and other calcium phosphates. TTCP is theonly calcium phosphate with Ca/P ratio above 1.67. Thus, this substancecan be mixed with other calcium phosphates with lower Ca/P ratio toobtain PHA or CDHA without the formation of acids or bases asby-products. Theoretically, any calcium phosphate more acid than PHA canreact directly with TTCP to form HA or CDHA according to the followingchemical reactions.

7Ca₄(PO₄)₂O+2Ca(H₂PO₄)₂·H₂O→6Ca₅(PO₄)₃OH+3H₂O   (7)

2Ca4(PO₄)₂O+Ca(H₂PO₄)₂·H₂ 0→<Ca₉(HPO₄) (PO₄)₅OH+2H₂O   (8)

Ca₄(PO₄)₂O+CaHPO₄·2H₂O→Ca₅(PO₄)₃OH+2H₂O   (9)

3Ca₄(PO₄)₂O +6CaHPO₄·2H₂O→2Ca₉(HPO₄) (PO₄)₅OH+13H₂O   (10)

Ca₄(PO₄)₂O+CaHPO₄→Ca₅ (PO₄)₃OH   (11)

3Ca₄(PO₄)₂O+6CaHPO₄→2Ca₉(HPO₄) (PO₄)₅OH +H₂O   (12)

3Ca₄(PO₄)₂O+Ca₉H₂(PO₄)₆·5H₂O→4Ca₅(PO₄)₃OH+4H₂O   (13)

3Ca₄(PO₄)₂O+3Ca₉H₂(PO₄)₆·5H₂O→4Ca₉(HPO₄) (PO₄)₅OH+14H₂O   (14)

Ca4(PO₄)₂O+2Ca₃(PO₄)₂+H₂O→Ca₅(PO₄)₃OH   (15)

In equations (7) and (8) DCPD is formed as an intermediate reactionproduct, but with PHA or CDHA at the end of the reaction. Reactions(13), (14), and (15) are all very slow. However, by using the formulas(9)-(12) it is possible to produce a cement which sets and hardens withtime at room or body temperature and at a neutral pH.

It is also possible to form PHA as the final hardened product by usingmixtures of calcium phosphates with a Ca/P ratio of less than 1.67. Thisis accomplished by using additional calcium sources, such as Ca(OH)₂ orCaCO₃, instead of TTCP. One example is the reactionβ-TCP+DCPD+CaCO₃→PHA. Initially formed crystals of PHA from a reactionbetween CDPD and CaCO₃ function as binders between β-TCP particles. WhenDCPD is consumed the reaction continues between the remaining calciumcarbonate and β-TCP with the formation of PHA. However, it seems thatthe latter process has a detrimental effect on the mechanical strengthof the cement.

It is preferred that the calcium phosphate with the capability of beinghardened to a calcium phosphate cement when reacting with an aqueousliquid is tricalcium phosphate (TCP), tetracalcium phosphate (TTCP),anhydrous dicalcium phosphate, monocalcium phosphate monohydrate (MCPM),dicalcium phosphate dihydrate (DCPD), or octocalcium phosphate (OCP).Preferably, the calcium phosphate is a-tricalcium phosphate.

In order to confer an initial strength to a bone mineral substitutematerial the calcium sulphate hemihydrate in the composition accordingto the invention should comprise 2-80 wt %, preferably 10-30 wt % of thedry powder to be mixed with an aqueous liquid. Likewise, the calciumphosphate to be hardened to a calcium phosphate cement should comprise10-98 wt %, preferably 70-90 wt % of the dry powder. In the composition,the aqueous liquid should comprise between 0.1 and 2 ml, preferablybetween 0.5 and 1 ml per gram powder.

By preferably using particulate reaction components in the inventivecomposition, a high strength implant material will be obtainedinitially. The fast setting calcium sulphate material will be formedwithin a block of a slow setting material, i.e. the calcium phosphatecement. Thus, when initial strength decreases the second strengthincreases, and its final strength will be maintained within the body.Pores, holes and cavities will gradually be formed as the sulphatedegrades, which acts like lacuna, and the finally set and hardenedimplant of a high strength material will look like a normal bone.

Both reactions in the inventive composition can be controlled byincluding an accelerator or a retarder. By using seed particles, theprocesses can be accelerated.

If such an accelerator is added, the calcium sulphate hemihydrate willset rapidly, i.e. within 10 min. Particulate calcium sulphate dihydrateis a suitable accelerator for this reaction, the particle size beingless than 1 mm. A more efficient reaction is obtained if the particulatecalcium sulphate dihydrate has a particle size of less than 150 μm,preferably less than 100 μm, and most preferable less than 50 μm. Theparticulate calcium sulphate dehydrate should comprise between 0.1 and10 wt %, preferably between 0.1 and 2 wt % of the calcium sulphatehemihydrate which is to react with an aqueous liquid. The acceleratorshould be adapted so that a set material is obtained within 15 min,preferably within 8 min, which has a threshold strength of about 30 MPain a clinical situation. Preferably, the particulate calcium sulphatedihydrate is α-calcium sulphate dehydrate.

The second reaction of a calcium phosphate to a calcium phosphate cementsets slowly, but can be controlled to set within 18 h as a bone mineralsubstitute material with a strength of about 30 MPa. This can beaccomplished by adding hardened particulate calcium phosphate cement tothe inventive composition. The hardened calcium phosphate cement can behydroxylapatite (HA), preferably precipitated hydroxylapatite (PHA),tricalcium phosphate (TCP), or a mixture thereof. It should have a Ca/Pratio between 1.5 and 2. The particulate calcium phosphate cement shouldhave a particle size which is less than 20 μm, preferably less than 10μm and comprise between 0.1 and 10 wt %, preferably between 0.5 and 5 wt% of the calcium phosphate which is to react with an aqueous liquid.

The reaction of calcium phosphate to a calcium phosphate cement can alsobe accelerated by a phosphate salt, for example disodium hydrogenphosphate (Na₂HPO₄), which is dissolved in the aqueous liquid. In thiscase, the accelerator should be present in the aqueous liquid atconcentrations of 0.1-10 wt %, preferably 1-5 wt %.

The two types of accelerator for the reaction of calcium phosphate tocalcium phosphate cement can be used either separately or incombination.

In the composition according to the invention the aqueous liquid can bedistilled water or a balanced salt solution, such as PBS, PBSS, GBSS,EBSS, HBSS, or SBF.

The injectability of the composition according to the invention can beimproved in several ways. It has surprisingly been shown that a pHreducing component can be added to the, inventive composition, theinjectability thereof being improved. Such a pH reducing component isfor example ascorbic acid or citric acid. These acids are included inthe sterile liquid or the sterile powder of the composition in amountsof 0.1-5 wt %, preferably 0.5-2 wt %.

Another way to improve the injectability of the composition is to add abiologically compatible oil. The concentration of the oil should bebetween 0.1 and 5 wt %, preferably between 0.5 and 2 wt %. A suitableoil to be used in the inventive composition is vitamin E. The oil caneither be intermixed with the sterile powder or included in the sterileliquid of the composition.

As stated above, the addition of a small amount of already reactedcalcium sulphate dihydrate had an effect on the injectable time of thebone mineral substitute. Thus, by replacing some of the non-reactedcalcium sulphate hemihydrate with reacted calcium sulphate dihydrate,the injectability of the composition could be improved. As much as 95%of the hemihydrate can be replaced. Preferably, 50-90% of thehemihydrate is replaced by the dihydrate, most preferred 80-90%.

In order to further improve the bone mineral substitute materialobtained with the inventive composition it is possible to furtherinclude additional substances, e.g. growth factors, anti-cancersubstances, antioxidants and/or antibiotics, etc. Antibiotic containingbone cement is already known and it has been shown that addition ofantibiotics to synthetic hydroxylapatite and cancellous bone releasessaid antibiotics in a concentration sufficient for treating boneinfections when said substances are administered into the bone.

An efficient mixing system must be available in order to prepare thecomposition according to the invention. The mixing can take place in aconventional cement mixing system and the composition is injected bymeans of a convenient delivery system. The mixing container ispreferably of that type which can suck the aqueous component into thepowder component (German Patent 4409610). This Prepack™ system is aclosed mixing system for delivery in combination with prepackedcomponents in a flexible foil bag. Other mixing devices can of coursealso be used, for example two interconnected soft bags which can beadapted to a delivering cylinder.

The formation of air bubbles in the composition, which can interferewith the hardening reaction of the calcium sulphate hemihydrate andresult in a decreased initial mechanical strength of the implantedmaterial during surgery, can be prevented by mixing the compositionunder conditions of subatmospheric pressure, e.g. in vacuo. However, anatmospheric pressure can also be used. Preferably, the powder componentof the composition is sterilized by means of radiation before it ismixed with the sterile liquid component.

EXAMPLES

The invention will now be further described and illustrated by,reference to the following examples. It should be noted, however, thatthese examples should not be construed as limiting the invention in anyway.

Comparative Example 1

As a control test the injectable time and the setting time of purecalcium sulphate hemihydrate were determined to be more than 10 and 20minutes, respectively.

Comparative Example 2

As a second control test the injectable time and the setting time of amixture of calcium sulphate hemihydrate, and hydroxylapatite were alsodetermined to be more than 10 and 20 minutes, respectively.

Comparative Example 3

The injectable time (IT) and the setting time (SI) were studied for thefirst reaction of a calcium sulphate hemihydrate to calcium sulphatedihydrate in the presence of a passive additive. Twenty differentmixtures of calcium sulphate hemihydrate, hydroxylapatite (HA) andaccelerator (Acc) were evaluated, which had different ratios ofhydroxylapatite and accelerator, see Table 3. The setting time wasdetermined by a mechanical test. A metallic rod having a weight of 23 g,a diameter of 10 mm and a length of 35 mm was dropped from a height of35 mm. The time when the rod did not leave any mark on the sample wasregistered as the setting time.

TABLE 3 TEST CASO₄ HA HA ACC IT SI NO. (G) (G) (%) (%) (MIN) (MIN) 1 324 10 10 1.5 3.0 2 28 8 20 10 1.5 4.0 3 24 12 30 10 1.5 4.0 4 20 16 40 102.0 6.0 5 16 20 50 10 1.5 6.0 6 34 4 10 5 2.0 5.0 7 30 8 20 5 1.5 5.0 826 12 30 5 2.5 7.0 9 22 16 40 5 2.5 7.5 10 18 20 50 5 2.0 7.0 11 35 4 102.5 1.5 5.0 12 31 8 20 2.5 1.5 5.0 13 27 12 30 2.5 2.0 7.5 14 23 16 402.5 2.5 7.5 15 19 20 50 2.5 2.5 10.0 16 35.6 4 10 1 2.5 7.0 17 31.6 8 201 3.0 9.0 18 27.6 12 30 1 3.5 10.5 19 23.6 16 40 1 4.0 13.0 20 19.6 2050 1 4.0 14.5

Example 1

Different bi-phasic injectable cements were produced, which were basedon a-tricalcium phosphate and α-calcium sulphate hemihydrate.

The mechanical strength of each cement produced was evaluated with timeat 10 hours, 24 hours, 3 days, and 14 days after mixing of the cementwith water. The evaluation was performed at the time periods given bymeans of a cylindrical specimen (d=6 mm, h=12 mm) that had been immersedin a physiological saline solution of 37° C. The results are shown inTable 4 below.

TABLE 4 Amount of Compressive Compressive Compressive Compressive α-TCPstrength 10 h strength 24 h strength 3 d strength 14 d (wt %) (MPa) ±S.D. (MPa) ± S.D. (MPa) ± S.D. (MPa) ± S.D. 0 11 3.63 7.64 1.41 12.992.66 9.66 3.2 20 1.01 0.39 1.69 0.49 3.99 0.35 5.36 0.33 40 0.68 0.255.08 1.66 8.82 1.2 9.82 1.86 60 3.58 1.02 5.1 0.91 15.73 5.24 14.13 1.4280 5.31 1.03 10.72 0.69 21.8 3.41 23.92 3.06 100 6.24 1.48 22.37 6.3437.99 4.74 33.98 10.37

Example 2

The compressive strength was further tested with reference to a-TCPcontaining less than 20 wt % calcium sulphate hemihydrate (CSH). (CSHwas obtained from Bo Ehrlander A B, Gothenborg, Sweden.)

The two powders were mixed together mechanically during 5 min. Then, theliquid was added to the powder at a liquid to powder (L/P) ratio of 0.32ml·g⁻ 1. The liquid contained 2.5 wt % Na₂HPO₄ as an accelerator.

Moulds were then filled and immersed in a saline solution (0.9%,) at 37°C. for 7 days. The results are shown in Table 5 below and in FIG. 1.

As seen in FIG. 1, the compressive strength was drastically increasedwhen the α-TCP content exceeded 80 wt %.

TABLE 5 Content of Compressive Standard No. of CSH (wt %) strength (MPa)Deviation (MPa) samples tested 0 62.62 7.98 7 5 34.60 9.65 7 10 23.5410.37 8 15 22.45 5.12 10

Example 3

During each of the two setting reactions, crystals are formed whencalcium sulphate hemihydrate and calcium phosphate, respectively, reactwith water in the setting reactions. Initially, crystal nuclei arecreated and the final crystal structure is then formed by growth fromthe nuclei. By adding already formed crystals of set material, thenucleation step in the setting process is already completed, which willdecrease the time needed to crystallize the material and make it hard.The crystals will grow directly from particles of added calcium sulphatedihydrate and hydroxylapatite, respectively. Thus, these added particlesof set material will act as accelerators in the setting reactions.

The smaller size of accelerator particles added to the material, themore efficient accelerating effect will be obtained because the crystalswill grow from the surface of the particles. If the acceleratorparticles are small, then the surface of the particles will be large perunit of weight.

When α-CaSO₄·2H₂O is used as an accelerator it will be more efficientthan β-CaSO₄·½H₂O, when α-CaSO₂·½H₂0 is used as the main component ofthe material. This could be explained by the crystal shape differencebetween the two forms of the calcium sulphate. Since the crystals aregrowing directly from the particle surface of the accelerator, thereaction proceeds faster if the accelerator crystals have exactly thesame shape as the crystals that are forming from the main component ofthe material.

Example 4

The effects of the content of calcium sulphate dihydrate on theinjection time is shown in FIG. 2. In this case the liquid/powder (L/P)ratio is 0.4 ml/g. The limit of injection time was defined when the loadreached N, which is comparable to the highest force by hand at whichinjection was possible.

Example 5

The effects of the water content and the content of calcium sulphatedihydrate on the setting time is shown in FIG. 3, wherein L/P is theliquid-powder ratio (ml/g). The setting time was measured by usingGillmore Needles according to ASTM Standard C266.

Example 6

In the inventive composition, the form of the calcium sulphatehemihydrate is of importance. α-Calcium sulphate hemihydrate(α-CaSO₄·½H₂O) is advantageous to use because of its mechanicalstrength. α-CaSO₄·½H₂O has a compressive strength of 40.4 MPa comparedwith 14 MPa for β-α-CaSO₄·½H₂O.

Example 7

Biodegradation of the Calcium Sulphate with Hydroxylapatite bonesubstitute in vitro and in vivo.

The degradation rate of calcium sulphate with 40 wt % hydroxylapatitewas investigated. The material was placed in a Simulated Body Fluid aswell as muscle pockets in rats. The mechanical strength and size of theblock obtained were investigated with time as a biodegradation index.

Mechanical testing

Compressive strength testing was performed using an MTS and Instron8511.20 testing equipment. After harvesting the materials, the sampleswere directly placed between self-levelling platens and compressed at 1mm min⁻¹ until failure at room temperature.

Volume measurements

After the material harvesting, a caliper measured the volume of theblock of material.

In vitro Study

Cements of calcium sulphate or calcium sulphate with hydroxylapatitewere prepared by mixing with distilled water at L/P ratio of 0.25 ml/g.After mixing the cement was injected into a PFTE mould and allowed toset. The samples were 4 mm in diameter and 8 mm in length. Sixcylindrical samples were placed in a Simulated Body Fluid, and theliquid was changed every day. After one week the samples were directlyplaced between self-levelling platens and subjected to compressivestrength testing until failure at room temperature.

In vivo Study

Materials preparation

Calcium sulphate hemihydrate (CaSO₄·½H₂O) was mixed with 40 wt %hydroxylapatite powder (Ca₁₀(PO₄)₆ (OH)₂; HA). The mixture of POP-HA wassintered and quenched in air. An accelerator (a calcium sulphate) wasadded at 0.4 wt % to the POP-HA, and the dry powder material wassterilized by gamma-irradiation.

A cement was prepared by mixing the powder with distilled water at a L/Pratio of 0.25 ml/g. Materials were prepared, which contained calciumsulphate or calcium sulphate+hydroxylapatite. After mixing, the cementwas injected into a PFTE mould and allowed to set. The samples werecylindrical with diameter of 4 mm and height of 8 mm. Once set, thesamples are inserted into muscle pockets of rats.

Animals

ague-Dawley rats weighing around 200 g were used and kept in animalfacilities for 1 week before use. The animals were fed a standardlaboratory diet. All rats were anesthetized with peritoneal injectionsof 0.5-0.6 ml of a solution containing 1 ml pentobarbital (60 mg/ml), 2ml diazepam (5 mg/ml), and 1 ml saline (0.15 M). The implants wereinserted in muscles of the rats. Nine rats were used for each periodstudied. The rats were killed by a peritoneal injection of an overdoseof pentobarbital at 1 or 4 weeks after implantation.

Results

After one week of incubation the mechanical strength was recorded of thecylindrical samples placed in the Simulated Body Fluid or musclespockets in rats, respectively. The mechanical strength of the materialshad decreased from 35 Mpa to about 5 Mpa both in vitro as well as invivo. The volume of remaining block was only ⅓ to 1/10 of the originalblock volume (Table 5).

After 4 weeks of incubation, the mechanical strength of the materialshad totally disappeared, and the rods of calcium sulphate were almostcompletely absorbed. The calcium sulphate with hydroxylapatite was stillpresent but totally deformed, and the material was surrounded by normalsoft tissue. The tissue also penetrated into the materials. Furthermore,the mass of remaining material was larger than the original blockimplanted.

Table 6 below shows the volume of remaining cylinder material (Mean ±SE)in rat muscles after an incubation of 1 or 4 weeks. The original volumeof the cylinder material was 100 mm³. Statistic analysis was performedby using the one way ANOVA method and Student's t-test. All resultsobtained exhibited a high statistical significance (p<0.0001).

TABLE 6 1 week incubation 4 weeks incubation No. of Volume No. of VolumeMaterial samples (mm³) samples (mm³) PoP 9 31.7 ± 3.1  9  1.9 ± 1.5 PoPt HA 9 6.1 ± 1.5 8 159.4 ± 21.7 PoP + HA + Vitamin E 8 9.1 ± 2.0 8 196.0± 17.9

The implanted material comprising calcium sulphate and hydroxylapatitewas rapidly degraded within one week in both Simulated Body Fluid and inrats. The rate of degradation was the same in Simulated Body Fluid ormuscles pockets, indicating that only one method is needed in order todemonstrate the degradation rate.

In conclusion, tests of the combined sulphate and phosphate materialexhibit biodegradation in vitro and in vivo as well as hardening of bothcomponents with good results with reference to injectability andsetting.

1-33. (canceled)
 34. A composition for a bone mineral substitutematerial comprising a dry powder to be mixed with an aqueous liquid, thedry powder comprising a first setting reaction component, which is acalcium sulphate hemihydrate; a second setting reaction component, whichis a calcium phosphate; and at least one accelerator for the settingreaction of both the first and the second setting reaction componentswith the aqueous liquid, wherein the at least one accelerator for thereaction of the second component with the aqueous liquid is dissolved inthe aqueous liquid.
 35. The composition of claim 34, wherein the calciumsulphate hemihydrate is an α-calcium sulphate hemihydrate.
 36. Thecomposition according to claim 34, wherein the calcium phosphate is atricalcium phosphate.
 37. The composition according to claim 34, whereinthe accelerator is disodium hydrogen phosphate (Na₂HPO₄).
 38. Thecomposition according to claim 34, wherein the accelerator comprises 0.1wt % to 10 wt % of the aqueous liquid.
 39. The composition according toclaim 38, wherein the accelerator comprises 1 wt % to 5 wt % of theaqueous liquid.
 40. The composition according to claim 34, wherein theaqueous liquid comprises distilled water or a balanced salt solution.41. The composition of claim 34, wherein the aqueous liquid comprisesbetween 0.1 ml and 2 ml per gram of the powder.
 42. The composition ofclaim 41, wherein the aqueous liquid comprises between 0.5 ml and 1 mlper gram of the powder.
 43. The composition of claim 34 furthercomprising a biologically active substance.
 44. The composition of claim43, wherein the biologically active substance is chosen from a growthfactor, an anti-cancer substance, an antibiotic, and an antioxidant, andmixtures thereof.
 45. A composition for a bone mineral substitutematerial comprising a dry powder to be mixed with an aqueous liquid, thedry powder comprising a first setting reaction component, which is acalcium sulphate hemihydrate; a second setting reaction component, whichis a calcium phosphate; at least one accelerator for the settingreaction of the first or the second setting reaction component, or boththe first and the second setting reaction components with the aqueousliquid, wherein the at least one accelerator for the reaction of thesecond component with the aqueous liquid is dissolved in the aqueousliquid, and a biologically compatible oil to improve the injectabilityof the composition.
 46. The composition of claim 45, wherein the calciumsulphate hemihydrate is an α-calcium sulphate hemihydrate.
 47. Thecomposition according to claim 45, wherein the calcium phosphate is atricalcium phosphate.
 48. The composition according to claim 45, whereinthe biologically compatible oil is vitamin E.
 49. The compositionaccording to claim 45, wherein the biologically compatible oil comprises0.1 wt % to 5 wt % of the composition.
 50. The composition according toclaim 49, wherein the biologically compatible oil comprises 0.5 wt % to2 wt % of the composition.
 51. A composition for a bone mineralsubstitute material comprising a dry powder to be mixed with an aqueousliquid, the dry powder comprising a first setting reaction component,which is a calcium sulphate hemihydrate; a second setting reactioncomponent, which is a calcium phosphate; at least one accelerator forthe setting reaction of the first or the second setting reactioncomponent, or both the first and the second setting reaction componentswith the aqueous liquid, wherein the at least one accelerator for thereaction of the second component with the aqueous liquid is dissolved inthe aqueous liquid; and a pH reducing component in order to improve theinjectability of the composition.
 52. The composition of claim 51,wherein the calcium sulphate hemihydrate is an α-calcium sulphatehemihydrate.
 53. The composition of claim 51, wherein the calciumphosphate is a tricalcium phosphate.
 54. The composition of claim 51,wherein the pH reducing component is ascorbic acid or citric acid. 55.The composition of claim 51, wherein the pH reducing component comprises0.1 wt % to 5 wt %.
 56. The composition of claim 55, wherein the pHreducing component comprises 0.5 wt % to 2 wt % of the composition. 57.A method of producing an injectable bone mineral substitute material,wherein the composition of claim 34 is mixed with the aqueous liquid ina closed mixing and delivery system.
 58. The method of claim 57, whereinthe mixing is conducted under conditions of subatmospheric pressure. 59.A method of producing an injectable bone mineral substitute material,wherein the composition of claim 51 is mixed with the aqueous liquid ina closed mixing and delivery system.
 60. The method of claim 59, whereinthe mixing is conducted under conditions of subatmospheric pressure. 61.A method of producing a composition for bone mineral substitutematerial, comprising contacting the composition of claim 34 with anaqueous liquid.
 62. The method of claim 61, wherein the composition iscapable of being hardened in a body fluid in vivo to a bi-phasic cementimplant that with time obtains a porous structure for bone ingrowth. 63.A kit for use in preparing a composition for a bone mineral substitutematerial, said kit comprising: (1) the composition according to claim34; and (2) optionally an aqueous liquid.